Activity of a novel protonophore against methicillin-resistant Staphylococcus aureus

Abstract

Aim: Compound 1-(4-chlorophenyl)-4,4,4-trifluoro-3-hydroxy-2-buten-1-one (compound 1) was identified as a hit against methicillin-resistant Staphylococcus aureus (MRSA) strain MW2.

Methods & results: The MIC of compound 1 against MRSA was 4 μg/ml. The compound showed enhanced activity at acidic pH by lowering bacterial intracellular pH and exhibited no lysis of human red blood cells at up to 64 μg/ml and its IC₅₀ against HepG2 cells was 32 μg/ml. The compound reduced 1-log₁₀ colony forming units of intracellular MRSA in macrophages and prolonged the survival of MRSA-infected Caenorhabditis elegans (p = 0.0015) and Galleria mellonella (p = 0.0002).

Conclusion: Compound 1 is a protonophore with potent in vitro and in vivo activity against MRSA and no toxicity in mammalian cells up to 8 μg/ml that warrants further investigation as a novel antibacterial.

Keywords: Galleria mellonella; MRSA infection; S. aureus; antibiotic; macrophages; protonophore.

Figure 1.. Structure of 1-(4-chlorophenyl)-4,4,4-trifluoro-3-hydroxy-2-buten-1-one (compound 1).

Figure 2.. In vitro killing of methicillin-resistant Staphylococcus aureus MW2 cells by compound 1.

(A) Time to kill assays: MRSA MW2 (10⁸ cells/ml) were treated with DMSO or compound 1 (16 μg/ml, 4× MIC) or gentamicin (8 μg/ml, 4× MIC) and CFUs enumerated after incubating for various times. (B) Killing of MRSA MW2 in RAW 264.7 macrophages. Macrophages were exposed to MRSA-MW2 cells (MOI = 25) for 2 h. Extracellular bacteria were killed by treatment with gentamicin (200 μg/ml) for 2 h and the macrophages exposed to 2× MIC compound 1 (8 μg/ml), 2× MIC vancomycin (8 μg/ml) or DMSO control for 20 h. Cells were lysed and the bacteria were plated and counted. Data represent the mean ± SD (n = 3). CFU: Colony forming unit; MOI: Multiplicity of infection; MRSA: Methicillin-resistant Staphylococcus aureus.

Figure 3.. Effect of pH on antibacterial activity.

The MICs of compound 1, vancomycin and gentamicin were measured against MRSA MW2 at pH: (A) 5.0, (B) 6.0, (C) 7.0 and (D) 8.0. Data represent the mean ± SD (n = 3). MRSA: Methicillin-resistant Staphylococcus aureus.

Figure 4.. Intracellular pH and membrane permeability.

(A) Staphylococcus aureus intracellular pH: MRSA-MW2 cells preloaded with the fluorescence pH indicator probe AM-BCECF were treated with 10 μg/ml of compound 1, vancomycin, CCCP or DMSO (negative control) and fluorescence measurements taken. (B) Membrane permeabilization by compound 1: MRSA-MW2 cells were loaded with the membrane impermeable nucleic acid stain Sytox green and treated with compound 1 (3.125–50 μg/ml) and the fluorescence was measured at regular time interval post treatment. Data represent the mean ± SD (n = 3). AM-BCECF: Acetoxymethyl ester, 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein; CCCP: Carbonyl cyanide m-chlorophenylhydrazone; MRSA: Methicillin-resistant Staphylococcus aureus.

Figure 5.. Hemolysis and cytotoxicity assays.

(A) Hemolytic activity of compound 1: human erythrocytes were treated with various concentrations of compound 1 (0.06–64 μg/ml) or Triton X-100 (0.001 to 1.0%) for 1 h and the supernatant was examined for hemolysis. (B) Cytotoxicity of compound 1 in HepG2 cells: HepG2 cells were treated with increasing concentrations of compound 1 (0.125–64 μg/ml) and incubated for 24, 48 or 72 h. Data represent the mean ± SD (n = 3).

Figure 6.. Whole animal Staphylococcus aureus infection models.

(A) Bright field and Sytox orange stained images of MRSA-MW2 infected Caenorhabditis elegans in the presence of DMSO (1%), compound 1 (2.86 μg/ml) or vancomycin (10 μg/ml). Images were obtained from the original HTS campaign. (B) Rescue of C. elegans nematodes infected with MRSA MW2 by compound 1 over the concentration range of 0.5–64 μg/ml. Data represent the mean ± SD (n = 15). (C) Galleria mellonella survival assay: Compound 1 (25 mg/kg) was administrated into the last right proleg before (prophylactic) or after (therapeutic) bacterial inoculation (2 × 10⁶ cells/ml). Vancomycin (25 mg/kg) and DMSO or no injection were used as positive and negative controls, respectively. HTS: High-throughput screening; MRSA: Methicillin-resistant Staphylococcus aureus.